This application claims priority under 35 U.S.C. xc2xa7xc2xa7119 and/or 365 to EP 98 40 1722,8 filed, in Europe on Jul. 7, 1998 and EP 98 40,2825,8 filed in Europe on Nov. 13, 1998; the entire contents of which are hereby incorporated by reference.
The present invention relates to a recombinant adenoviral vector deleted of all or part of the E1 region and a part of the E4 region but retaining sufficient E4 sequences to improve expression and/or persistence of expression of a recombinant gene in a host cell or organism. Furthermore, it relates to the use of adenoviral E4 open reading frames (ORFs) to improve expression or persistence of expression of a recombinant gene inserted in an expression vector. Finally, the invention relates to a method for preparing a viral particle, a cell, a pharmaceutical composition comprising such vectors as well as their therapeutic or prophylactic use. The invention is of very special interest in relation to prospect for gene therapy, in particular in men.
Gene therapy can be defined as the transfer of genetic material into a cell or an organism to treat or prevent a genetic or acquired disease. The possibility of treating human disorders by gene therapy has changed in a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the USA in September 1990 on a patient who was genetically immunodeficient as a result of a mutation affecting the gene encoding adenine deaminase (ADA). The relative success of this first experiment encouraged the development of this technology for various genetic and acquired diseases. The large majority of the current protocols employ vectors to carry the therapeutic gene to the cells to be treated. Numerous viral or synthetic vectors have been developed during these last years. Their structure, organization and biology are described in the literature available to a person skilled in the art.
Adenoviruses have been detected in many animal species, are nonintegrative and not very pathogenic. They are able to infect a variety of cell types, dividing as well as quiescient cells. They have a natural tropism for airway epithelia. In addition, they have been used as live enteric vaccines for many years with an excellent safety profile. Finally, they can be easily grown and purified in large quantities. These features have made adenoviruses particularly appropriate for use as gene therapy vectors for therapeutic and vaccine purposes. Their genome consists of a linear double-standed DNA molecule of approximately 36 kb carrying more than about thirty genes necessary to complete the viral cycle. The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4) which contain 6 transcription units directed by their own promoters. The E1, E2 and E4 regions are essential for viral replication whereas the E3 region, which is believed to modulate the anti-viral host immune response, is dispensable for viral growth in vitro. The late genes (L1 to L5) encode in their majority the structural proteins constituting the viral capsid. They overlap at least in part with the early transcription units and are transcribed from a unique promoter (MLP for Major Late Promoter). In addition, the adenoviral genome carries at both extremities cis-acting regions essential for DNA replication. These are the 5xe2x80x2 and 3xe2x80x2 ITR (Inverted Terminal Repeat) and a packaging sequence following 5xe2x80x2 ITR.
The E4 region is believed to be involved in viral DNA replication, late mRNA synthesis, viral assembly and the shut off of host protein synthesis. It is a complex transcription unit which encodes a variety of polypeptides. Those encoded by the open reading frames (ORFs) 6 and 7 are assumed to compete with the cellular RB protein for the binding to the E2F transcription factor, confering a function of transactivators. The expression product of ORF4 is able to bind and regulate the cellular phosphatase 2A to modulate the activity of viral (E1A) and cellular transcription factors. The polypeptides encoded by ORFs 3 and 6 are essential to viral growth because of their capability to maturate the primary 28 kb transcript derived from the adenoviral genome or its export into the cytoplasm. Their absence might be complemented in trans to allow the viral growth. In addition, the ORF6 polypeptide interacts with the E1B encoded 55K polypeptide to form a complex that facilitates the cytoplasmic accumulation of late messengers at the expense of cellular mRNA.
The adenoviral vectors presently used in gene therapy protocols lack most of the E1 region in order to avoid their dissemination in the environment and the host body. Additional deletions in the E3 region allow to increase the cloning capacity. The gene of interest is introduced into the viral DNA in place of a deleted region. The feasibility of gene transfer using these vectors designated xe2x80x9cfirst generationxe2x80x9d has been demonstrated in a number of cases. However, the question of their safety is still under evaluation. Indeed, the probability to generate replication-competent viruses during their propagation in conventional complementing cell lines, is not negligible. Furthermore, the potential immunogenicity of viral proteins still expressed by the viral backbone may reduce the persistence of transduced cells as well as the long term expression of the recombinant transgene and may be associated with inflammatory events.
These major drawbacks have led to the construction of vectors of second generation that retain the cis regions necessary for viral replication (ITRs and packaging sequences) and contain substantial genetic modifications aimed to abolish the residual synthesis of the viral antigens which is postulated to be responsible for the stimulation of inflammatory responses (see for example the international application WO94/28152 or U.S. Pat. No. 5,670,488 which discloses adenoviral vectors partially deleted of E4 sequences with the exception of ORF3 or ORF6/7 that do not need E4 complementation). A minimal vector deficient for the whole adenoviral functions can also be considered.
The persistence of transgene expression is a prerequisite before envisaging the general use of adenoviral vectors in human gene therapy protocols, in particular in view of treatment of chronic and genetic diseases. However, deletion of the E4 region has been recently shown to alter transgene expression conducted by a heterologous promoter (i.e. CMV promoter, RSV LTR). Coinfection studies indicated that E4 products could be supplied in trans to restore stable transgene expression (Armentano et al., J. Virol. 71 (1997) 2408-2416; Brough et al., J. Virol. 71 (1997), 9206-9213).
Thus, the technical problem underlying the present invention is the provision of expression vectors which do not show the instability of transgene expression as observed in E4-deleted adenovirus vectors and of means which allow to obtain long term expression of a transgene in cells.
This problem is solved by the provision of the embodiments characterized in the claims.
Accordingly, the present invention relates to a recombinant adenoviral vector derived from an adenovirus genome in which at least all or part of the E1 region is deleted or non-functional and a part of the E4 region has been deleted and which comprises a gene of interest operably linked to regulatory elements, wherein said adenoviral vector retains sufficient E4 sequences to improve expression and/or persistence of expression of said gene of interest in a host cell or organism. Preferably, the retained E4 sequences consist of:
(i) ORF3 and ORF6+ORF7;
(ii) ORF3 and ORF7;
(iii) ORF3 and ORF6;
(iv) ORF3 and ORF6/7;
(v) ORF3 and ORF4; or
(vi) ORFs 1, 2, 3 and 4.
It was surprisingly found that impaired transgene expression in E4-deleted adenoviral vectors could be fully restored by the presence and expression of certain E4 ORFs, in particular of the above-mentioned E4 ORFs.
Although the E4 region may vary between the different adenovirus strains, it can be identified on the basis of nucleotide sequences available in different sources (publications or data bank) or by homology with the well characterized Ad5 E4 region. As an indication, the E4 region is located at the right end of the adenoviral genome, with the E4 promoter being localized 5xe2x80x2 to 3xe2x80x2 ITR. Transcription occurs from right to left with regards to the adenoviral map. The E4 region reveals 7 open reading frames of which 6 contain an AUG start codon (ORFs 1, 2, 3, 4, 6 and 7) and codes for at least 6 polypeptides (the ORF7 encoded protein has not yet been identified) which can be identified by sequence analysis. mRNA mapping studies have also identified two new ORFs created by mRNA splicing events, i.e. ORF3/4 and ORF6/7. In particular, in the Ad5 genome ORF6 extends from nt 34074 to 33192, ORF7 extends from nt 33111 to 32913 and ORF6/7 extends from nt 34074 to 33901 (splice donor) and 33189 (splice acceptor) to 32913 (see e.g. Cutt et al., J. Virol. 61 (1987), 543-552; Freyer et al., Nucl. Acids Res. 12 (1984), 3503-3519; Virtanen et al., J. Virol. 51 (1984), 822-831). Thus, a recombinant adenoviral vector according to the invention may, inter alia, retain the ORF6, the ORF7 and/or the ORF6/7. A construct retaining ORF6, ORF7 and ORF6/7 contains both ORF6 and ORF7 which can lead to produce the corresponding mRNAs and the splice product ORF6/7. Such a sequence is called ORF6+7 in the scope of the present invention. Furthermore, the recombinant adenoviral vector according to the present invention can also comprise ORF3 and ORF4, which includes the combinations ORF3 and ORF3/4, ORF3/4 and ORF4, ORF3 and ORF4 or just ORF3/4. The person skilled in the art is able to modify the precited E4 region of an adenoviral genome by conventional molecular biology techniques in order to obtain an E4 region which retains the above-mentioned ORFs and lacks the remaining sequences. In particular, it is well within the reach of the person skilled in the art to delete from an adenoviral E4 region a specific portion of DNA, e.g. by appropriate restriction or endonuclease digest and religation. Another possibility is to isolate the retained E4 sequences by PCR.
It is possible for the person skilled in the art to determine those ORFs present in the E4 region which exert a positive effect on transgene expression, e.g. by deleting these ORFs from the E4 region and determining whether they affect transgene expression. Furthermore, it is possible to test the effect of an E4 ORF by providing it in cis or trans to a E4 deleted vector carrying a transgene and determining its effect on transgene expression. Such methods are provided in the examples. The E4 ORF(s) retained in the adenoviral vector is (are) capable alone or in combination, directly or by means of other cellular or viral factors to improve the expression of a gene of interest inserted into the adenoviral vector. This positive effect on transgene expression may be exerted at different levels: transcription, elongation, transport, stability of the transgene mRNA or alternatively translation. The improvement is determined by evaluation of the transgene expression product or persistence of its expression in in vivo or in vitro experiments.
The adenoviral vectors according to the invention moreover may show a reduced hepatotoxicity in comparison to vectors comprising the complete E4 region.
Preferably, the recombinant adenoviral vectors retains the entire coding sequences of one or more of the above-mentioned E4 ORF(s) extending from the initiator ATG to the stop codon. However, it is also feasible to employ a functional variant having promoter regulation capacities. Such a variant may be obtained by mutation or truncation of the native ORF sequences. To illustrate this embodiment, one may refer to an E4 ORF6 variant deleted of the sequence comprised between the first and the second ATG codon. More preferably, the E4 ORFs retained in the adenoviral vector comprise regulatory elements allowing their expression, more preferably their natural regulatory elements, in particular the E4 promoter. Alternatively, they can also be operably linked to a heterologous promoter. In order to stabilize expression of the E4 ORFs, it may be advantageous that the E4 ORF(s) retains or comprises splicing sequences. They may be homologous (to the E4 sequences) or heterologous (i.e., derived from any eukaryotic gene or of synthetic origin). In principle all splicing sequences described in the prior art are suitable, e.g. those of the genes encoding xcex1 or xcex2 globin, apolipoprotein, immunoglobulin, factor IX, factor VIII, CFTR or of the pCI vector (Promega). The E4 ORFs retained in the adenoviral vector according to the invention may be those naturally occurring in such a vector. In particular, they may remain at their natural location. However, it is also possible that the vector is constructed by deleting all E4 sequences, in particular all E4 ORFs, and inserting certain E4 ORFs from the same or other adenovirus backbones in the adenoviral vector at a location where the E4 region normally resides or at a different location, e.g. in place of the deleted E1 or E3 region. The ORFs may be oriented in sense or antisense orientation with respect to the direction of transcription of the wild-type E4 region.
The adenoviral vector according to the invention is an adenoviral vector in which at least all or part of the E1 region is deleted or non-functional. Such a vector can be derived from an adenovirus genome in which at least all or part of the E1 region is deleted. It can be obtained from a parent adenovirus whose genome has been modified. The modifications may be diverse (deletion, mutation and/or addition of one or more nucleotides) and concern any viral sequence. In addition, they may be localized in the coding sequences of the viral genome or outside of these sequences, for example in the regulatory elements such as promoters. As a guide, some viral sequences may be deleted, rendered non functional or replaced by heterologous nucleotide sequences and, in particular gene(s) whose expression is sought in a host cell (one or more gene of interest).
Preferably, the vector according to the invention is defective for E1 functions by total or partial deletion of the respective region. The deletion encompasses at least E1a sequences and may extend in the E1b transcription unit. Preferably the E1b sequences overlapping with pIX gene are not deleted. Such E1 deletions are included in prior art vectors published in the literature.
It goes without saying that the adenoviral backbone of the vector according to the invention may comprise additional modifications, such as deletions, insertions or mutations in one or more viral genes. According to an advantageous embodiment, it is derived from an adenovirus genome in which one or more viral genes of the E2 and/or L1-L5 regions is non-functional. The non functionality may be obtained by a partial or complete deletion or by mutation of one or more of the cited regions. As an example, one may refer to the thermosensible mutation located on the DBP (DNA Binding Protein) encoding gene of the E2a region (Ensinger et al., J. Virol. 10 (1972), 328-339). A defective adenoviral vector deficient in all early and late regions may also be envisaged.
In a further preferred embodiment the vector is deleted of all or part of the E3 region. In this context, it might be interesting to retain the E3 sequences coding for the polypeptides allowing to escape the host immune system, in particular those coding for gpl9k glycoprotein (Gooding et al., Critical Review of Immunology 10 (1990), 53-71).
The adenoviral vector according to the present invention may be derived from a human or animal adenovirus genome, in particular of canine, avian, bovine, murine, ovine, feline, porcine or simian origin or alternatively from a hybrid thereof Any serotype can be employed, in particular the murine adenovirus Mav1 (Beard et al., Virology 175 (1990), 81-90), the canine CAV-1 or CAV-2 (Spibey et Cavanagh, J. Gen. Virol. 70 (1989), 165-172; Linne, Virus Research 23 (1992), 119-133; Shibata et al., Virol. 172 (1989), 460-467; Jouvenne et al., Gene 60 (1987), 21-28), avian DAV (Zakharchuk et al., Arch. Virol. 128 (1993), 171-176) or the bovine BAV3 (Mittal et al., J. Gen. Virol. 76 (1995), 93-102). However, the human adenoviruses of C sub-group are preferred and especially adenoviruses 2 (Ad2) and 5 (Ad5). Generally speaking, the cited viruses are available in collections such as ATCC and have been the subject of numerous publications describing their sequence, organization and biology, allowing the artisan to practice them. For example, the sequence of the human adenovirus type 5 is disclosed in the Genebank data base under the reference M 73260 and is incorporated by reference in its entirety.
As mentioned before, it was found that certain ORFs of the E4 region are capable of regulating positively the expression of a gene of interest inserted in an adenoviral vector. The term xe2x80x9cgenexe2x80x9d refers to a nucleic acid (DNA, RNA or other polynucleotide derivatives) which can be of any origin (prokaryote, eukaryote, viral . . . ). The gene of interest can code, e.g., for an antisense RNA, a ribozyme or a messenger (mRNA) that will be translated into a protein of interest. It includes genomic DNA, cDNA or mixed types (minigene). It may code for a mature polypeptide, a precursor (i.e. precursor intended to be secreted and comprising a signal sequence, a precursor intended to be maturated by proteolytic cleavage . . . ), a fragment of a protein (truncated protein), a chimeric polypeptide originating from the fusion of diverse sequences or a mutated polypeptide displaying improved and/or modified biological properties. The gene may be isolated from any organism or cell by the conventional techniques of molecular biology (PCR, cloning with appropriate probes, chemical synthesis) and if needed its sequence may be modified by mutagenesis, PCR or any other protocol.
The following genes of interest are of particular interest. They may code for a cytokine (xcex1, xcex2 or xcex3 interferon, interleukine (IL), in particular IL-2, IL-6, IL-10 or IL-12, a tumor necrosis factor (TNF), a colony stimulating factor GM-CSF, C-CSF, M-CSF . . . ), a cell or nuclear receptor, a receptor ligand (fas ligand), a coagulation factor (FVIII, FIX . . . ), a growth factor (FGF stating for Fibroblast Growth Factor, VEGF stating for Vascular Endothelial Growth Factor), an enzyme (urease, renin, thrombin, metalloproteinase, nitric oxide synthase NOS, SOD, catalase . . . ), an enzyme inhibitor (xcex11-antitrypsine, antithrombine III, viral protease inhibitor, PAI-1 which stands for plasminogen activator inhibitor), the CFTR protein, insulin, dystrophin, a MHC antigen (Major Histocompatibility Complex) of class I or II or a polypeptide that can modulate/regulate expression of correponding genes, a polypeptide capable of inhibiting a bacterial, parasitic or viral infection or its development (antigenic polypeptides, antigenic epitopes, transdominant variants inhibiting the action of a native protein by competition . . . .), an apoptosis inducer or inhibitor (Bax, Bc2, Bc1 X . . . ), a cytostatic agent (p21, p 16, Rb . . . ), an apolipoprotein (ApoAI, ApoAIV, ApoE . . . ), an angiogenesis inhibitor (angiostatin, endostatin . . . ), an oxygen radical scaveyer, a polypeptide having an anti-tumor effect, an antibody, a toxin, an immunotoxin and a marker (xcex2-galactosidase, luciferase . . . ) or any other genes of interest that are recognized in the art as being useful for the treatment or prevention of a clinical condition.
For example, in view of treating an hereditary dysfunction, one may use a functional copy of a defective gene, for example a gene encoding factor VIII ou IX in the context of haemophilia A or B, dystrophin (or minidystrophin) in the context of myopathies, insulin in the context of diabetes, CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) in the context of cystic fibrosis. Suitable genes of interest to delay or inhibit tumor or cancer progression, include but are not limited to those encoding an antisense RNA, a ribozyme, a cytotoxic product such as thymidine kinase of herpes-1 simplex virus (TK-HSV-1), ricin, a bacterial toxin, the expression product of yeast genes FCY1 and/or FUR1 having UPRTase (Uracile Phosphoribosyl Transferase) and CDase (Cytosine Desaminase) activities, an antibody, a polypeptide inhibiting cellular division or transduction signals, a tumor suppressor gene (p53, Rb, p73. . . ), a polypeptide activating host immune system, a tumor-associated antigen (MUC-1, BRCA-1, an HPV early or late antigen (E6, E7, L1, L2. . . ) . . .), optionally in combination with a cytokine gene. Finally, in the context of anti-HIV therapy, one may use a gene encoding an immunoprotective polypeptide, an antigenic epitope, an antibody (2F5; Buchacher et al., Vaccines 92 (1992), 191-195), the extracellular domain of CD4 (sCD4; Traunecker et al., Nature 331 (1988), 84-86), an immunoadhesine (i.e. CD4-IgG hybrid, CD4-2F5 fusion; Capon et al., Nature 337 (1989), 525-531; Byrn et al., Nature 344 (1990), 667-670), an immunotoxin (i.e. resulting from fusion between angiogenine and 2F5 or CD4-2F5; Kurachi et al., Biochemistry 24 (1985), 5494-5499), a trans-dominant variant (EP 0614980, W095/16780), a cytotoxic product (see above) or IFNxcex1 or xcex2.
In addition, a gene of interest may also comprise a selection gene allowing the selection of transfected and transduced cells. Such genes include but are not limited to the neo gene (encoding neomycin phosphotransferase) confering resistance to G418, dhfr (Dihydrofolate Reductase), CAT (Chloramphenicol Acetyl transferase), pac (Puromycine Acetyl-Transferase) and gpt (Xanthine Guanine Phosphoribosyl Transferase). Selection genes are known in the art.
The gene of interest may be engineered as a functional expression cassette, including a suitable promoter. It may be obtained from any viral, bacterial or eukaryotic gene (even from the gene of interest), be constitutive or regulable. Cptionally, it may be modified in order to improve its transcriptional activity, delete negative sequences, modify its regulation, introduce appropriate restriction sites etc. Suitable promoters include but are not limited to the followings: adenoviral E1a, MLP, PGK (Phospho Glycerate Kinase), MT (metallothioneine; Mc Ivor et al., Mol. Cell Biol. 7 (1987), 838-848), xcex1-1 antitrypsin, CFTR, surfactant, immunoglobulin, xcex2-actin (Tabin et al., Mol. Cell Biol. 2 (1982), 426-436), SRxcex1 (Takebe et al., Mol. Cell. Biol. 8 (1988), 466-472), early SV40 (Simian Virus), RSV (Rous Sarcoma Virus) LTR, TK-HSV-1, SM22 (WO 97/38974), Desmin (WO 96/26284) and early CMV (Cytomegalovinis). Alternatively, the promoter may be stimulated in tumor or cancerous cells. As an example, one may employ the promoters isolated from MUC-1 gene overexpressed in breast and prostate cancers (Chen et al., J. Clin. Invest. 96 (1995), 2775-2782), CEA (Carcinoma Embryonic Antigen) overexpressed in colon cancers (Schrewe et al., Mol. Cell. Biol. 10 (1990), 2738-2748), tyrosinose overexpressed in melanomas (Vile et al., Cancer Res. 53 (1993), 3860-3864), ERB-2 overexpressed in breast and pancreas cancers (Harris et al., Gene Therapy 1 (1994), 170-175) and xcex1-foetoprotein overexpressed in liver cancers (Kanai et al., Cancer Res. 57 (1997), 461-465). The early CMV promoter is preferred in the context of the invention.
The regulatory elements may further include additional elements, such as intron(s), secretion signal, nuclear localization signal, IRES, poly A transcription termination sequences, tripartite leader sequence and replication origins.
The adenoviral vector according to the present invention may comprise one or more gene(s) of interest. The different genes may be included in the same cassette or in different cassettes thus controled by separate regulatory elements. The cassettes may be inserted into various sites within the vector in the same or opposite directions.
In another aspect the present invention also relates to the use of a polynucleotide comprising one or more open reading frames (ORFs) of the E4 region of an adenovirus selected from the group consisting of ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6/7, ORF6 and ORF7, either alone or in combination, to improve the expression and/or persistence of expression of a gene of interest which is contained in an expression vector and is operably linked to regulatory elements.
The finding that the presence of certain ORFs of the adenoviral E4 region is advantageous for the stable expression of transgenes is also of importance for obtaining stable expression of transgenes in expression vectors others than adenoviral vectors. Thus, the above-mentioned ORFs of an adenoviral E4 region can generally be used to achieve stable expression of transgenes in expression systems. In this regard, it is possible to achieve improved expression, i.e. expression at all or stable long term expression, of a transgene either by providing the ORFs in cis or in trans.
As indicated above in connection with the adenoviral vectors according to the invention, the E4 region may vary between the different adenovirus strains. However, it is well within the skill of the person skilled in the art to identify the E4 region of an adenovirus as well as the ORFs contained in it. Thus, it is possible for the person skilled in the art to isolate the above-cited ORFs from an adenoviral genome in order to use them according to the invention.
The E4 ORF(s) used in the scope of the present invention may be from any adenoviral origin (animal or human). Preferably they are derived from a human adenovirus of sub-group C, particularly preferred from Ad2 or Ad5.
The E4 ORF(s) is (are) capable alone or in combination, directly or by means of other cellular or viral factors to improve the expression of a gene of interest inserted into an expression vector. This positive effect on transgene expression may be exerted at different levels: transcription, elongation, transport, stability of the transgene mRNA or alternatively translation. The improvement is determined by evaluation of the transgene expression product or persistence of its expression in in vivo or in vitro experiments. One way to proceed is to inject a vector carrying said E4 ORF(s) together with an expression cassette of a gene whose product is easily detectable (LacZ, luciferase, xcex11-antitrypsin, factor IX, CFTR . . . ) and to determine the level of transgene product over time compared to a control that is devoided of E4 adenoviral sequences. The improvement can be seen in terms of the amount of transgene product (at least by a factor 2) or in terms of persistence of the expression (stability over a longer period of time).
In a preferred embodiment of the use according to the invention, a polynucleotide is used which comprises
(i) ORFs 1, 2, 3 and 4;
(ii) ORFs3,6+7;
(iii) ORFs 3 and 7;
(iv) ORFs 3 and 6;
(v) ORF 3 and ORF 6/7;
(vi) ORFs 3 and 4; or
(vii) ORF 3/4
of an adenoviral E4 region.
Preferably, such an ORF comprises the complete coding sequence, i.e. from the initiator ATG to the stop codon. However, it is also possible to employ a functional variant of such an ORF, e.g. variants obtained by deletion, mutation or truncation which still encode a functional polypeptide. Variants include in particular such ORFs which share a high degree of homology to the native equivalent, in particular at least 70% sequence identity, more preferably at least 80% and even more preferred at least 90%. Particularly preferred is absolute identity.
The polynucleotide used in the present invention is operably linked to regulatory elements to allow its expression in a host cell or organism. Such elements include a promoter that may be isolated from any gene of eukaryotic or viral origin. When the polynucleotide comprises several E4 ORFs, these may be expressed from a unique promoter or independent ones. In this case, the different E4 cassettes may be in the same or opposite direction and in the same and different locations within one or more vector(s). However, the use of a unique promoter to drive transcription of the selected E4 sequences is preferred. A first possibility is to place them under the control of the homologous E4 promoter. Another alternative is to use a heterologous promoter. Such a heterologous promoter is preferably constitutive and may be chosen among those cited hereinafter. The person skilled in the art is capable to link said polynucleotide to an appropriate promoter in an operative way. In order to stabilize expression, it may be advantageous that the E4 ORF(s) retains or comprises splicing sequences. They may be homologous (derived from E4 sequences) or heterologous (derived from any eukaryotic gene or from synthetic origin). The large variety of splicing sequences described in the state of the art are suitable in the context of the invention. One may cite more particularly those isolated from genes encoding xcex1 or xcex2 globin, apolipoprotein, immunoglobulin, factor IX, factor VIII, CFTR and from the pCI vector (Promega).
Advantageously, the polynucleotide sequence is inserted in an expression vector. In the context of the present invention, it can be a plasmid, a synthetic or a viral vector. Plasmid denotes an extrachromosomic circular DNA capable of autonomous replication in a given cell. The choice of the plasmids is very large. It is preferably designed for amplification in bacteria and expression in eukaryotic host cell. Such plasmids can be purchased from a variety of manufactors. Suitable plasmids include but are not limited to those derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene), pCI (Promega) and p Poly (Lathe et al., Gene 57 (1987), 193-201). It is also possible to engineer such a plasmid by molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.). A plasmid may also comprise a selection gene in order to select or identify the transfected cells (by complementation of a cell auxotrophy, antibiotic resistance), stabilizing elements (i.e. cer sequence; Summers and Sherrat, Cell 36 (1984), 1097-1103) or integrative elements (LTR viral sequences).
An expression vector may also be from viral origin and may be derived from a variety of viruses, such as herpes viruses, cytomegaloviruses, AAV (adeno-associated virus), poxviruses (canarypox, fowlpox, vaccinia virus, MVA) and retroviruses.
In a preferred embodiment the polynucleotide and gene of interest are inserted into the same expression vector. They may be inserted in the same location (i.e. in place of the deleted E1 sequences in an E1xe2x88x92 adenoviral vector) or at different locations (e.g., the gene of interest in place of the deleted E1 sequences and the polynucleotide in place of the native E4 region). The use of two independant expression vectors each carrying said polynucleotide and gene of interest is also feasible. In this case, both vectors may be introduced in the host cell together (co-transfection or co-infection) or separately.
In a particularly preferred embodiment the vector into which the polynucleotide comprising the E4 ORFs are inserted, is an adenoviral vector, preferably one from which the E4 region has been deleted.
With respect to the nature and structure of the adenoviral vector, the location of the inserted E4 ORFs, the nature of the gene of interest and the regulatory regions, the same applies as already set forth in connection with the adenoviral vectors according to the invention.
Moreover, the present invention relates to a non-adenoviral vector comprising a gene of interest operably linked to regulatory elements and comprising one or more open reading frames (ORF(s)) of the E4 region of an adenovirus selected from the group consisting of ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6/7, ORF6 and ORF7 taken individually or in combination and operably linked to regulatory elements.
With respect to the combinations and characterisitics of the ORFs and the nature of the gene of interest the same applies as set forth above for the use of a polynucleotide according to the invention.
The present invention also relates to an infectious viral particle comprising an adenoviral vector according to the invention or an expression vector comprising E4 ORFs as described in connection with the use according to the invention. It may be prepared according to any conventional technique in the field of the art. When an adenoviral vector is considered, one may proceed by cotransfection of suitable adenoviral fragments in a cell line such as 293 line (as described in Graham and Prevect, Methods in Molecular Biology, Vol 7 (1991), Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc, Clinton, N.J.). It is also possible to reconstitute the vector in Escherichia coli by ligation or homologous recombination (as described in WO96/17070) before transfecting the cell line. Furthermore, the virions may be amplified by passage in a permissive cell in order to generate a high titer viral stock that may be used in the preparation of clinical lots. It may be propagated in a complementation cell line, which supplies in trans the deleted/mutated viral functions. Line 293, established from human embryonic kidney cells (Graham et al., J. Gen. Virol. 36 (1977), 59-72) is commonly used to complement the E1 function. Other cell lines have been engineered to complement doubly defective vectors (E1xc2x0 E4xc2x0 or E1xc2x0 E2xc2x0), such as those described in Yeh et al. (J. Virol. 70 (1996), 559-565), Krougliak and Graham (Human Gene Therapy 6 (1995), 1575-1586), Wang et al. (Gene Therapy 2 (1995), 775-783), Lusky et al. (J. Virol. 72 (1998), 2022-2033) and in the international applications WO94/28152 and WO97/04119. Alternatively, it is also possible to also use a helper virus supplying in trans at least a part of the viral deficiencies.
The invention also relates to a method for preparing an infectious viral particle according to the invention, according to which:
(i) the adenoviral vector of the invention or the expression vector comprising E4 ORFs as described in connection with use according to the the present invention is introduced into a complementation cell capable of complementing in trans said vector, to obtain a transfected complementation cell;
(ii) said transfected complementation cell is cultured under suitable conditions to permit the production of said infectious viral particle; and
(iii) said infectious viral particle is recovered from the cell culture.
The vector can be introduced into the cell line by any one of a variety of methods known in the art. One may proceed by transfection of the vector or fragments thereof, by lipofection, electroporation and infection. The infectious viral particles may be recovered from the culture supernatant but also from the cells which can be lysed, for example, by a series of thawing/freezing cycles. Optionally, the virions may be amplified and purified according to standard techniques (chromatography, ultracentrifugation, for example in a cesium chloride gradient . . . ).
Furthermore, the present invention relates to a host cell comprising an adenoviral vector of the invention or a polynucleotide and an expression vector as defined in connection with the use of the invention or infected by an infectious viral particle of the invention. Preferably, such a cell is from mammalian origin and, in particular from human origin. The vector may be inserted into the cellular genome or not (episome). Suitable cells include but are not limited to primary or tumoral cells, from haematopoietic (totipotent stem cell, leucocyte, lymphocyte, monocyte, macrophage . . . ), muscular (satellite cell, myocyte, myoblaste, smooth muscle cells . . . ), cardiac, lung, tracheal, liver, vascular, epithelial, fibroblastic or endothelial origin.
The present invention also relates to a composition, preferably a pharmaceutical composition, comprising as an agent an adenoviral vector according to the invention, a polynucleotide and an expression vector as described in connection with the use of the invention, a host cell or an infectious viral particle according to the invention or prepared according to the process of the invention. The composition of the invention is intended especially for the preventive or curative treatment of disorders such as genetic diseases (haemophilia, diabete, cystic fibrosis, Duchenne or Becker myopathies, auto-immune diseases) or acquired disorders (cancers, tumors, cardiovascular diseases, viral diseases such as AIDS or hepatitis B or C . . . ).
The composition according to the invention may be manufactured in a conventional manner for local, general or oral administration. Aerosol, intillation or injection may be envisaged. Suitable routes of administration include intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraarterial, intraperitoneal, intratumoral, intranasal, intrapulmonary or endotracheal routes. The administration may take place in a single dose or a dose repeated one or several times after a certain time interval. The appropriate administration route and dosage vary in accordance with various parameters, for example, with the individual, the disorder to be treated or with the gene of interest to be transferred. The viral particles according to the invention may be formulated in the form of doses of between 104 and 1014 iu(infectious unit), advantageously 105 and 1013 iu and preferably 106 and 1012 iu. The titer may be determined by conventional techniques (see for example Lusky et al., 1998, supra). Doses based on vector may comprise between 0.01 and 100 mg of DNA, advantageously 0.05 and 10 mg and preferably 0.5 and 5 mg. In addition, the agent of the composition may be combined with a vehicle which is acceptable from a pharmaceutical standpoint. The formulation may also include a diluent, an adjuvant or an excipient. It may be presented as a liquid directly injectable or as a dry powder (lyophylized . . . etc) that can be reconstituted before use.
In addition, the vector, host cell and viral particles according to the present invention can optionally be combined with one or more substances improving gene transfer efficiency or stability. Such substances are well known in the ,art (see for example Felgner et al. (Proc. West. Pharinacol. Soc. 32 (1987), 115-121); Hodgson and Solaiman (Nature Biotechnology 14 (1996), 339-342); Remy et al. (Bioconjugate Chemistry 5 (1994), 647-654)) and include in particular polymers, cationic lipids, liposomes, nuclear proteins and neutral lipids. They may also be used in combination (i.e. cationic and neutral lipids).
Moreover, the present invention relates to the use of an adenoviral vector according to the invention, a polynucleotide and an expression vector, as described in connection with the use according to the invention, a viral particle or a host cell according to the invention for the preparation of a drug intended for gene transfer into a host cell or organism and preferably for the treatment of human or animal body by gene therapy or immunotherapy. According to a first possibility, the drug may be administered directly in vivo (for example by intravenous injection, in an accessible tumor, in the lungs by aerosol . . . ). It is also possible to adopt the ex vivo approach which consists in removing cells from the patient (bone marrow stem cells, peripheral blood lymphocytes, muscle cells . . . ), transfecting or infecting them in vitro according to standard protocols and readministering them to the patient.
In a preferred embodiment of said use of the present invention the retained E4 sequences in said adenoviral vector are:
(i) ORFs 3 and 4;
(ii) ORF 3/4;
(iii) ORFs 3 and 6+7;
(iv) ORFs 3 and 6;
(v) ORFs 3 and 7; or
(vi) ORF 3 and 6/7.
Preferably, the corresponding pharmaceutical compositions are for gene transfer into lung tissue.
In another preferred embodiment of said use of the present invention the retained E4 sequences in said adnoviral vector are:
(i) ORF 3;
(ii) ORFs 3 and 4;
(iii) ORF 3/4;
(iv) ORFs 3 and 6+7;
(v) ORFs 3 and 6;
(vi) ORFs 3 and 7; or
(vii) ORFs 3 and 6/7.
Preferably, the corresponding pharmaceutical compositions are for gene transfer into liver tissue.
The adenoviral vectors mentioned in context with said preferred embodiments of the use of the present invention display a reduced toxicity and/or provoke a reduced inflammatory response by the host cell or host organism. This will be illustrated by the examples which will follow below.
The present invention also relates to a method of treatment according to which a therapeutically effective amount of an adenoviral vector according to the invention, a polynucleotide and an expression vector as described in connection with the use according to the invention, a viral particle or a host cell according to the invention is administered to a patient in need of such a treatment.
Finally, the invention also provides a product comprising:
(i) an expression vector comprising a gene of interest operably linked to regulatory elements; and
(ii) a polynucleotide as defined above in connection with the use according to the invention;
as a combination product for a simultaneous or separate use.
According to this embodiment, the expression vector is conventional and may be derived from a plasmid or a virus. The term vector encompasses the free genome (DNA) or the genome packaged in a viral capsid (virion). As indicated before, the polynucleotide may also be inserted in such a vector.
The constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning detailed in Sambrook et al. (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, N.Y., or more recent editions) or according to the manufacture""s recommendations when a commercial kit is used. Homologous recombination steps preferentially employ the strain E. Coli BJ 5183 (Hanahan, J. Mol. Biol. 166 (1983), 557-580) and are performed as described in Chartier et al. (J. Virol. 70 (1996), 4805-4810). Regarding the repair of restriction sites, the technique employed consists of filling in the 5xe2x80x2 protuding ends using the large fragment of E. coli DNA polymerase I (Klenow). The adenoviral genome fragments employed in the different constructions described below are indicated precisely according to their positions in the nucleotide sequence of the Ad5 genome as disclosed in the Genebank databank under the reference M73260.
Regarding the cell biology, the cells are transfected or transduced according to the standard techniques well known to a person skilled in the art. The calcium phosphate technique may be mentioned but any other protocol may also be employed. The culture conditions are, for their part, conventional. Line 293 (Graham et al, 1977, supra ; ATCC CRL-1573) results from the integration in its chromosomes of the 5xe2x80x2 end of the Ad5 genome. Line TG5606 (described in Lusky et al. (1998), supra) is derived from line 293 stably transfected by the plasmid pTG5606 carrying the Ad5 E4 ORF6+7 (i.e. a sequence containing both ORF6 and ORF7 susceptible to produce the corresponding polynucleotides and the splice product ORF6/7) sequences and the pac selectable gene. A549 is available at ATCC (CCL-125). Other cell lines may be used as well.
Virus generation, viral growth and titration. For the generation of viruses, the viral genomes were released from the respective recombinant plasmids by PacI digestion and transfected into the appropriate complementation cell lines, as described previously (Chartier et al., 1996, J. Virol 70, 4805-4810). Wild type E4 vectors were generated in 293 cells, whereas all E4-modified vectors were generated in TG5606 cells. Virus propagation, purification and titration of infectious units (IU/ml), by indirect DBP inimunofluorescence was exactly as described (Lusky et al, 1998, supra). Viral particle concentration (P/ml) of each vector preparation was calculated using the optical density for measurement of viral DNA content (Mittereder et al., 1996, J. Virol. 70, 7498-7509). Growth of E4-modified vectors in the presence and absence of E4 complementation was assessed in 293 and compared to that in TG5606 cells.
Animal studies. The mice used in this study were 6 to 8 weeks old female immunocompetent C57B1/6, CBA or immunodeficient C.B17-scid/scid mice (IFFA Credo, L""albresle, France). The vectors containing the CFTR transgene were administered intratracheally (I.T.) or intravenously (IV) at the indicated doses. Animals were sacrificed at the times indicated. Organs were removed, cut into equal pieces and immediately frozen in liquid nitrogen until analysis.
DNA analysis. Total DNA was extracted from tissue culture cells and organs as described (Lusky et al., 1998, supra). Briefly , the cells or tissues were digested overnight with proteinase K solution (1 mg proteinase K in 1% SDS) in DNA lysis buffer (10 mM Tris-HCL pH 7.4, 400 mM NaCl, 2 mM EDTA). The DNA was isolated by phenol-chloroforn extraction followed by ethanol precipitation. DNA (10 xcexcg) was digested with BamHI and analyzed by Southern blot analysis using a 32P-labeled EcoRI-HindIII restriction fragment purified from Ad5 genomic DNA (nt 27331 to 31993).
RNA analysis. Total RNA was extracted from tissue culture cells and organs as described and as recommended by the supplier. For Northern blot analysis, 10-15 xcexcg of total RNA was subjected to gel electrophoresis and transferred to nitrocellulose filters. CFTR-specific mRNA was detected using a 32P-labeled BamHI restriction fragment (2540 bp) purified from the CFTR cDNA. Virus specific mRNA was detected using a 32P-labeled oligonucleotide, specifically hybridizing to the hexon mRNA of the viral L3 messages.
Toxicity studies. Toxicity studies of the different adenoviral constructs were conducted on six weeks-old immunocompetent female mice (C57BL/6, Balb/c, C3H or CBA) purchased from Iffa Credo (L""albresles, France). Mice were housed in specific pathogen-free facilities. Adenoviral vectors were delivered via intravenous (i.v.) administration by tail vein infusion of a volume of 1005 xcexcl. Animals were sacrificed at different time points over a period of at least one month (5, 16 and 30 or 4, 14, 30 and 60 days). Livers were removed and stored in 10% formaldehyde buffer until anatomopathological analysis. Viral particles concentration was calculated using the optical density for measurement of viral DNA content (Mittereder et al., 1996, J. Virol. 70, 7498-7509).
Anatomopathological analysis was performed on 10% formalin-fixed, paraffin-embedded tissues stained with hematoxilin and eosin. Liver damages (dystrophy) and inflammation status (lymphocytic infiltration) were visually estimated.
Liver toxicity was evaluated by analysing transaminases contents from serum samples taken at the time of sacrifice. GOT (glutamic oxalacetic transaminase) and GPT (glutamic pyruvic transaminase) levels were measured by a standard enzymatic method. GOT is representative of aspartate aminotransferase (AST) activity which catalyses the transfer of an amino group between L-aspartate and 2-oxaloglutarate to give L-glutamate and oxaloacetate, the latter reacting with NADH in the presence of malate deshydrogenase. The NADH oxidation rate is evaluated as the reduction of optical absorbance at 340 nm (Cobas Integra) and is directly proportional to AST catalytic activity. GPT is representative of alanine aminotransferase (ALT) activity which catalyses the reaction between L-alanine and 2-oxoglutarate to give L-glutarnate and pyruvate, the latter reacting with NADH in the presence of lactate dehydrogenase. The NADH oxidation rate was evaluated as the reduction of optical absorbance at 340 nm and is directly proportional to ALT catalytic activity.